The dynamics of surface detachment and quorum sensing in spatially controlled biofilm colonies of Pseudomonas aeruginosa
Biofilms represent a highly successful life strategy of bacteria in a very broad range of environments and often have negative implications for industrial and clinical applications, as their removal from surfaces and the prevention of biofouling in the first place represent formidable and to date unmet challenges. At the same time, biofilms modulate important natural processes, including nutrient cycling in rivers and streams and the clogging of porous materials. Biofilm development is a dynamic process, dependent on a host of cellular and environmental parameters that include, among others, hydrodynamic environment and the communication among cells (QS). Here we used microfluidics and micro-contact printing, paired with video-microscopy and image analysis, to study several aspects relating to the temporal dynamics of bacterial biofilms. Beyond reporting on specific findings from these experiments, we demonstrate how these innovative technologies can aid in obtaining a new layer understanding on biofilm processes and biofilm removal, thanks to unprecedented control over the biofilm's microenvironment. In a first set of experiments (chapter 2), we cultured Pseudomonas aeruginosa biofilms in a microfluidic channel for different times, after which we used the passage of an air plug as a mechanical insult to drive detachment. We found that the adhesion properties of an early-stage biofilm have a strong correlation with the time of growth and that biofilm detachment can occur in a spatially heterogeneous manner characterized by a regular pattern of 'holes' in the original biofilm. The resulting spatial distribution of bacteria correlates with the distribution of the extra polymeric substance (EPS) matrix before the insult, indicating that the locations of the holes correspond to where there was the least EPS. These results demonstrate that the detachment mechanism is a competition between the shear force exerted by the external flow and the local adhesion force of a given patch of biofilm, in turn governed by the local amount of EPS. This mechanism, and the observed heterogeneity in the detachment, imply that even at rather high shear rates, where the bulk of the biofilm is removed, local strongholds survive detachment, and may represent seeds for the colony to reform. In a second set of experiments (chapter 3), we examined the effects of patch size and hydrodynamic environment on QS induction on spatially defined patches of Pseudomonas aeruginosa biofilm. We found that the smaller biofilm patches start QS earlier than those in the larger patches. However at later times the proportion of auto-induced bacteria (normalized by the surface area covered by the cells) is higher in the larger patches. We expanded on these findings by investigating the contribution of flow to QS induction on the patches. The effect of ambient fluid flow was to accelerate the induction of quorum sensing compared to static conditions at moderate flow rates, due to the increase in the convective supply of nutrients and to quench quorum sensing at high flow rates, due to the autoinducer signal being washed out by flow. These findings establish microfluidics as a new tool in the study of biofilms, which enables both accurate control over microenvironmental conditions and direct observation of the dynamics of biofilm formation and disruption. Chapter 4 deepens the exploration of the role of micro-spatial heterogeneity on microbial processes by presenting a numerical model of how heterogeneity and microbial behavior (chemotaxis) affect trophic in a microbial food web. Results show that the intensity of the trophic transfer strong depends on the motile behavior of the different trophic levels: trophic transfer is enhanced when directional motility towards resource patches outweighs the random component of motility inherent in all microbial locomotion. Finally, in the Appendices, we demonstrate how the methods developed in this thesis can help in the assessment of the antifouling capabilities of new-generation surfaces, designed to prevent fouling and in the assessment of the cell adhesion on surfaces, fabricated with an arrangement of spatially localized hydrophobic patterns. In summary, this thesis demonstrates that use of new micro-technology and associated mathematical modeling enables new insights into the colonial life form of microorganisms and may provide impetus for new approaches to prevent biofouling on surfaces or remove biofilms from surfaces.
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